Date of Award

1981

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Interdepartmental Program

Abstract

The purpose of this study was to determine the pharmacokinetics of calcium leucovorin in normal subjects and in patients undergoing High-Dose methotrexame therapy for the treatment of neoplastic disease. By characterizing the disposition of leucovorin it was hoped that the efficacy of the aethotrexate therapy might be improved, while at the same time, the incidence of toxicity could be diminished.

An assay for the separation and quantification of leucovorin in serum was developed. The reduced folate vas extracted from serum using reverse-phase chromatographic minicolumns and a paired-ion reagent. Leucovorin was eluted from this system in methanol and evaporated to dryness under nitrogen. The extracted samples were stable for 3 days when stored at -4°C. High pressure liquid chromatography vas used to separate leucovorin from the extracted serum components using a reverse-phase, paired-ion mode. Ammonium phosphate, dibasic, was found to be a suitable pairing agent. The recently developed radially compressed reverse-phase columns were found to provide superior resolution of serum components than vas possible with conventional stainless steel columns. The effects of flow rate, pH, column temperature, and buffer concentration on the separation of leucovorin were investigated.

Electrochemical detection vas used to quantify the reduced folate in the extracted serum samples. An applied potential of 0.8 volts vas used to produce a sensitive and selective means of detecting leucovorin. The apparent lower limit of sensitivity under the conditions employed was 12 ng leucovorin. The effects of pH, buffer concentration, methanol concentration, and mobile phase flow rate on detector response were investigated.

The protein binding of leucovorin and its major metabolite 5-aethyltetrahydrofolate to human serum albumin was determined. Over the concentration range of 5.0x10-7M to 1x10-4M neither folate saturated the available binding sites. Leucovorin binding was a constant 701, while aethyltetrahydrofolate decreased slightly over this range. When both folates were present in the albumin solution, there was a significant decrease in the degree of binding of each ftethotrexate did not affect the binding of leucovorin or its metabolite.

The pharmacokinetics of leucovorin vas determined in 6 normal subjects and in 5 cancer patient s. There were no significant differences in the kinetic parameters calculated from either group. The disposition of leucovorin is best characterized by a two compartment open model. In initial half-life of 8.79 ± 4.44 minutes and a second, slower elimination phase of 231.46 ± 31.76 minutes was determined. The volume of the central compartment was calculated as 5.49 ± 3.53 Liters. Leucovorin serum concentrations in 3 cancer patients receiving High- Dose methotrexate were determined to be 3 to 11 times greater than vas predicted using methotrexate pharmacokinetic parameters.

This study clearly demonstrates that there are significant differences in the pharmacokinetics of leucovorin and methotrexate. The data presented in this study suggests that it might be possible to reduce the amount of leucovorin administered to patients being treated with methotrexate. This may be expected to improve the efficacy of the antimetabolite therapy without additional risk of toxicity.

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